1995 Fiscal Year Final Research Report Summary
Isolation and characterization of the nuclear matrix in higher plant cells
| Project/Area Number |
05640738
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
植物生理
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| Research Institution | HOKKAIDO UNIVERSITY (1995)
Akita Prefectural College of Agriculture (1993-1994) |
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Principal Investigator |
MASUDA Kiyoshi Hokkaido Univ., Fac. of Agr., Assoc. Prof., 農学部, 助教授 (60157203)
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| Co-Investigator(Kenkyū-buntansha) |
ONO Michiyuki Akita Pref. Coll. Agr., Biotech. Inst., Lect., 生物工学研究所, 講師 (50201405)
INOUE Masayasu Akita Pref. Coll. Agr., Biotech. Inst., Prof., 生物工学研究所, 教授 (90176446)
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| Project Period (FY) |
1993 – 1995
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| Keywords | Nuclear matrix / Nuclear protein / Carrot (nuclear structure) / somatic embryo |
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| Research Abstract |
Nuclei were isolated from somatic embryos of carrot using a buffer system containing non-ionic detergent. To prepare nuclear matrices, the purified nuclei were digested with DNase, followed by extraction with a high concentration of NaCl. A monoclonal antibody, CML-1, raised against carrot nuclear-matrix proteins, selectively labeled the nuclear periphery of carrot protoplasts when visualized by confocal and electron microscopy. The antibody scarcely labeled the cytoplasm and nucleoplasm of interphase cells. CML-1 recognized several proteins with apparent molecular masses of 96-130 kDa that were urea-extracted from the residual nuclear matrices. To identify the constituent proteins of higher plant cells structurally homologous to the vertebrate nuclear lamina, wer cloned a cDNA encoding a CML-1-recognized protein from a lambdagt11 carrot embryo cDNA expression library and determined its sequence. The protein was designated NMCP1. When the deduced amino acid sequence wascompared with other known protein sequences contained in major databases, no protein was found to show high sequence identity across the whole region of NMCP1. However, the amino acid sequence showed partial strong similarities with myosin heavy chain, tropomyoshin and some intermediate filament proteins including vertebrate lamins. Structural analysis of the NMCP1 protein indicated that it contained a long alpha-helical region exhibiting haptad repeats of apolar residues, demonstrating significant structural similarity with those of filament-forming proteins. In addition, there were potential sequence motifs for nuclear locating signals and substrates for different types of protein kinases. These results suggest that NMCP1 protein forms coiled-coil filaments and is a constituent of the peripheral architecture of the higher plant cell nucleus.
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